Eukaryotic cells initiate a multifaceted response to DNA damage that includes the ubiquitination of key substrates, leading either to their degradation or to the modulation of their activity. Despite the importance of the ubiquitination machinery in facilitating this response, the identities of the ubiquitin ligases involved in this pathway and their key targets are just beginning to be discovered. This proposal seeks to utilize novel, high throughput protein identification technologies developed in the investigator's laboratory to identify the key enzymes and substrates involved in this process. Using a mass spectrometry-based approach, they will analyze the global ubiquitination pattern of proteins of S. cerevisiae, determine how this pattern is altered in response to DNA damaging agents, and identify the enzymes responsible for generating this ubiquitin-mediated DNA damage response. A better understanding of how cells employ the ubiquitination pathway to protect themselves from DNA damage will be critical in developing an understanding of how the inactivation of this process can facilitate tumorigenesis.